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Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells.

Mariani SM, Matiba B, Armandola EA, Krammer PH - J. Cell Biol. (1997)

Bottom Line: The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis.These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands.Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology, German Cancer Research Center, Heidelberg.

ABSTRACT
The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNF-family and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1beta-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO. These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

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Induction of apoptosis and DNA fragmentation in Ag8  cells by recombinant mouse TRAIL. (A) The human T leukemia  cells Jurkat (Jurkat) (left panels), the mouse myeloma cells Ag8  (Ag8) (central panels), and the mouse T hybridoma cells 2H11  (TcHy) (right panels) were incubated with SN from Sf9 cells expressing mouse TRAIL (TRAIL) (upper panels), or mouse wildtype CD95L (CD95L) (lower panels). Results are presented as  forward/side scatter analysis of cellular morphology. Apoptotic  cells are shown in gate R1. (B) Soluble DNA was extracted from  Ag8 (Ag8) (lanes 1–3) and Jurkat cells (Jurkat) (lanes 4–6) (0.5 ×  106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1  and 4) or from Sf9 cells expressing mouse CD95L (lanes 2 and 5)  or mouse TRAIL (lanes 3 and 6). Extracted DNA was separated  on an agarose gel and visualized by ethidium bromide staining.  Molecular mass markers (MM) are shown. No soluble DNA was  extracted from TRAIL-resistant T hybridoma cells incubated  with either ligand.
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Figure 3: Induction of apoptosis and DNA fragmentation in Ag8 cells by recombinant mouse TRAIL. (A) The human T leukemia cells Jurkat (Jurkat) (left panels), the mouse myeloma cells Ag8 (Ag8) (central panels), and the mouse T hybridoma cells 2H11 (TcHy) (right panels) were incubated with SN from Sf9 cells expressing mouse TRAIL (TRAIL) (upper panels), or mouse wildtype CD95L (CD95L) (lower panels). Results are presented as forward/side scatter analysis of cellular morphology. Apoptotic cells are shown in gate R1. (B) Soluble DNA was extracted from Ag8 (Ag8) (lanes 1–3) and Jurkat cells (Jurkat) (lanes 4–6) (0.5 × 106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1 and 4) or from Sf9 cells expressing mouse CD95L (lanes 2 and 5) or mouse TRAIL (lanes 3 and 6). Extracted DNA was separated on an agarose gel and visualized by ethidium bromide staining. Molecular mass markers (MM) are shown. No soluble DNA was extracted from TRAIL-resistant T hybridoma cells incubated with either ligand.

Mentions: Recombinant TRAIL was tested for functional activity by incubating SN from TRAIL-expressing Sf9 cells with the human leukemic cells Jurkat. TRAIL killed the target cells but no cell death was observed in Jurkat cells incubated with control medium or SN of Sf9 cells expressing mouse gld CD95L. (Fig. 2 A). To assess the ability of recombinant TRAIL to affect mouse cells, a similar assay was performed with the mouse myeloma cells Ag8 as target. As shown in Fig. 2 B, Ag8 cells were killed very efficiently by TRAIL but not by the control mouse gld CD95L. It is of note that the same target cells were almost completely resistant to recombinant mouse and human CD95L. Specificity was further indicated by the lack of cell death in T hybridoma cells incubated with mouse TRAIL and CD95L (Fig. 3 A).


Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells.

Mariani SM, Matiba B, Armandola EA, Krammer PH - J. Cell Biol. (1997)

Induction of apoptosis and DNA fragmentation in Ag8  cells by recombinant mouse TRAIL. (A) The human T leukemia  cells Jurkat (Jurkat) (left panels), the mouse myeloma cells Ag8  (Ag8) (central panels), and the mouse T hybridoma cells 2H11  (TcHy) (right panels) were incubated with SN from Sf9 cells expressing mouse TRAIL (TRAIL) (upper panels), or mouse wildtype CD95L (CD95L) (lower panels). Results are presented as  forward/side scatter analysis of cellular morphology. Apoptotic  cells are shown in gate R1. (B) Soluble DNA was extracted from  Ag8 (Ag8) (lanes 1–3) and Jurkat cells (Jurkat) (lanes 4–6) (0.5 ×  106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1  and 4) or from Sf9 cells expressing mouse CD95L (lanes 2 and 5)  or mouse TRAIL (lanes 3 and 6). Extracted DNA was separated  on an agarose gel and visualized by ethidium bromide staining.  Molecular mass markers (MM) are shown. No soluble DNA was  extracted from TRAIL-resistant T hybridoma cells incubated  with either ligand.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139852&req=5

Figure 3: Induction of apoptosis and DNA fragmentation in Ag8 cells by recombinant mouse TRAIL. (A) The human T leukemia cells Jurkat (Jurkat) (left panels), the mouse myeloma cells Ag8 (Ag8) (central panels), and the mouse T hybridoma cells 2H11 (TcHy) (right panels) were incubated with SN from Sf9 cells expressing mouse TRAIL (TRAIL) (upper panels), or mouse wildtype CD95L (CD95L) (lower panels). Results are presented as forward/side scatter analysis of cellular morphology. Apoptotic cells are shown in gate R1. (B) Soluble DNA was extracted from Ag8 (Ag8) (lanes 1–3) and Jurkat cells (Jurkat) (lanes 4–6) (0.5 × 106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1 and 4) or from Sf9 cells expressing mouse CD95L (lanes 2 and 5) or mouse TRAIL (lanes 3 and 6). Extracted DNA was separated on an agarose gel and visualized by ethidium bromide staining. Molecular mass markers (MM) are shown. No soluble DNA was extracted from TRAIL-resistant T hybridoma cells incubated with either ligand.
Mentions: Recombinant TRAIL was tested for functional activity by incubating SN from TRAIL-expressing Sf9 cells with the human leukemic cells Jurkat. TRAIL killed the target cells but no cell death was observed in Jurkat cells incubated with control medium or SN of Sf9 cells expressing mouse gld CD95L. (Fig. 2 A). To assess the ability of recombinant TRAIL to affect mouse cells, a similar assay was performed with the mouse myeloma cells Ag8 as target. As shown in Fig. 2 B, Ag8 cells were killed very efficiently by TRAIL but not by the control mouse gld CD95L. It is of note that the same target cells were almost completely resistant to recombinant mouse and human CD95L. Specificity was further indicated by the lack of cell death in T hybridoma cells incubated with mouse TRAIL and CD95L (Fig. 3 A).

Bottom Line: The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis.These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands.Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology, German Cancer Research Center, Heidelberg.

ABSTRACT
The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNF-family and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1beta-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO. These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

Show MeSH
Related in: MedlinePlus